Sequence-independent substrate selection by the eukaryotic wobble base deaminase ADAT2/3 involves multiple protein domains and distortion of the tRNA anticodon loop

The essential deamination of adenosine A₃₄ to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the first structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A novel gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A₃₄ deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.